FLASH conditional knockout ES cells.

(A) Generation of conditional FLASH knockout ES clones. FLASH^flox/- ES clones expressing MerCreMer were established as indicated. The activation of Cre recombinase was induced by treating cells with 4-OHT (4-hydroxytamoxifen). Arrows (number 1–4) indicate the position of the primers for genomic PCR and black boxes (Probe 1 and Probe 2) indicate the position of the probes for Southern blot analyses. Neo^r and DT-A show neomycin-resistant and diphtheria toxin-A genes, respectively. (B) Genomic PCR analysis with primers 1 and 2 or primers 3 and 4 was carried out for wild-type ES (WT) and FLASH^flox/- (f/-) ES clones, and showed that recombination at loxP sites by MerCreMer (MCM) was correctly induced by the treatment with 4-OHT. (C) A deficiency in the FLASH protein in FLASH conditional KO ES cells was confirmed by Western blot analysis with an anti-FLASH monoclonal antibody. (D) Cell growth was examined after inducing the knockout of FLASH with the 4-OHT treatment for the indicated days. (E) Embryoid body formation was analyzed after inducing FLASH knockout with the 4-OHT treatment. Embryoid bodies were generated using the hanging drop method, and observed after a 10-day cultivation. (F) The expression of the indicated linage markers was analyzed during embryoid body formation (EB) by RT-PCR.